Use of an adrenal hormone-modifying agent

ABSTRACT

The present invention relates to a method of treating a disease or disorder characterised by increased stress hormone levels and/or decreased androgen hormone levels in a subject, comprising administering to the subject a therapeutically effective amount of a compound represented by formula (I): 
     
       
         
         
             
             
         
       
     
     wherein n is 1 or 3; R is hydrogen or —C(O)N(R a )(R b ) wherein R a  and R b  are independently —(C 1 -C 4 )alkyl, or —(C 1 -C 4 )alkyl-(C 5 -C 7 )aryl, wherein each of R a  and R b  is optionally substituted by —(C 1 -C 4 )alkoxy; R 1 , R 2 , and R 3 , are independently hydrogen, halogen, cyano or —(C 6 -C 10 ) aryl, wherein said —(C 6 -C 10 )aryl is optionally substituted by halogen, with the proviso that no more than one of R 1 , R 2 , and R 3  is hydrogen; and R 4  and R 5  are hydrogen; or a pharmaceutically acceptable salt thereof.

FIELD OF INVENTION

The invention relates to the use of a compound with adrenalhormone-modifying properties in disease states characterised byincreased stress hormone levels and/or decreased androgen hormonelevels.

BACKGROUND OF THE INVENTION

Steroidogenesis in the adrenal gland occurs via highly related andcontrolled cytochrom P450 enzymes. Inhibition of Aldosterone Synthase orthe enzyme cytochrom P450 11B2 (CYP11B2) represents a newpharmacological strategy to reduce excessive aldosterone levels.Aldosterone is a mineralocorticoid that is mainly synthesized in theadrenal gland and released into the circulation to control in the renalepithelium the sodium/potassium balance and thus water homeostasis andblood pressure as well as the in non-epithelial tissue of heart andkidney the formation of extracellular matrix and organ remodeling.Aldosterone synthase mediates in the adrenal gland the terminal andrate-limiting conversion of 11-deoxycorticosterone to corticosterone via11-beta-hydroxylation, the conversion of corticosterone to18-hydroxy-corticosterone via 18-methylhydroxylation and finally theconversion of 18-hydroxy-corticosterone to aldosterone via18-methyloxidation. The activity and expression of the enzyme is mainlyregulated by angiotensin II, potassium and adrenocorticotropin. Theseregulators of aldosterone synthase are sensitive to the actions ofaldosterone and the physiological circadian rhythm and as such create anendocrine feedback loop. Angiotensin II is produced upon stimulation ofrenin activity that is triggered via sodium loss and blood pressuredecrease due to hypoaldosteronemic states. Potassium is retained inexchange to sodium loss in hypoaldosteronemic conditions. Finally,adrenocorticotropin is produced from the pituitary gland in response tolow glucocorticoid levels and the circadian rhythm. Hence, a selectiveinhibition of aldosterone synthase and a reduction of aldosteronesecretion is counteracted with the stimulation of renin and thegeneration of angiotensin II as well as by a retention of potassium;both increased angiotensin II and potassium levels being potentstimulators of aldosterone synthase activity and thus aldosteronesecretion. The circadian rhythm upon aldosterone synthase inhibition isblunted for aldosterone yet adrenocorticotropin levels are notsignificantly changed as glucocorticoids are the main regulators ofadrenocorticotropin secretion. The rate-limiting enzyme of cortisolsecretion is the adrenal enzyme 11-beta-hydroxylase or cytochrom P45011B1 (CYP11B1) that converts 11-deoxycortisol to cortisol. Cortisollevels are controlled via the hypothalamic-pituitary-adrenal feedbackloop by controlling the release of adrenocorticotropin (ACTH).Adrenocorticoptropin stimulates in the adrenal gland the early and latesteroidogenic reactions leading to the synthesis of cortisol but alsodehydroepiandrosterone and androstendione (see FIG. 1, a diagram foradrenal steroidogenesis). The cortisol-producing enzyme CYP11B1 shows ahigh sequence homology of 95% at the amino acid level to thealdosterone-producing enzyme CYP11B2. Therefore, a compound targeted ataldosterone synthase to reduce excessive aldosterone secretion needs tobe tested for its enzyme selectivity.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of treating adisease or disorder characterised by increased stress hormone levelsand/or decreased androgen hormone levels in a subject, comprisingadministering to the subject a therapeutically effective amount of acompound represented by formula (I) or a pharmaceutically acceptablesalt thereof.

In another aspect, there is provided a method of treating heart failure,cachexia, acute coronary syndrome, chronic stress syndrome, cushing'ssyndrome or metabolic syndrome, comprising administering to the subjecta therapeutically effective amount of a compound represented by formula(I) or a pharmaceutically acceptable salt thereof.

In a further aspect, there is provided the use of a compound of formula(I) or a pharmaceutically acceptable salt thereof, for the preparationof a pharmaceutical composition for the treatment of a disorder ordisease characterised by increased stress hormone levels and/ordecreased androgen hormone levels in a subject

In a further aspect, there is provided the use of a compound of formula(I) or a pharmaceutically acceptable salt thereof, in the treatment of adisorder or disease characterised by increased stress hormone levelsand/or decreased androgen hormone levels in a subject.

In another aspect, the present invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, for thepreparation of a pharmaceutical composition for the treatment of adisorder or disease selected from heart failure, cachexia, acutecoronary syndrome, chronic stress syndrome, cushing's syndrome ormetabolic syndrome.

In a further aspect, there is provided the use of a compound of formula(I) or a pharmaceutically acceptable salt thereof, in the treatment of adisorder or disease selected from heart failure, cachexia, acutecoronary syndrome, chronic stress syndrome, cushing's syndrome ormetabolic syndrome.

DETAILED DESCRIPTION OF THE INVENTION

The compounds that can be used in the present invention is described byusing the following formula (I)

wherein n is 1 or 3;R is hydrogen or —C(O)N(R_(a))(R_(b)) wherein R_(a) and R_(b) areindependently —(C₁-C₄)alkyl, or —(C₁-C₄)alkyl-(C₅-C₇)aryl, wherein eachof R_(a) and R_(b) is optionally substituted by —(C₁-C₄) alkoxy;R₁, R₂, and R₃, are independently hydrogen, halogen, cyano or—(C₆-C₁₀)aryl, wherein said —(C₆-C₁₀)aryl is optionally substituted byhalogen, with the proviso that no more than one of R₁, R₂, and R₃ ishydrogen; andR₄ and R₅ are hydrogen; or a pharmaceutically acceptable salt thereof.

In one embodiment the compound of Formula (I) is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitrilehaving formula (II).

As used herein, the term “alkyl” refers to a fully saturated branched orunbranched hydrocarbon moiety. Preferably the alkyl comprises 1 to 6carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbonatoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms. Representativeexamples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl and the like.

As used herein, the term “alkoxy” refers to alkyl-O—, wherein alkyl isdefined herein above. Representative examples of alkoxy include, but arenot limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy- andthe like. As used herein, the term “lower alkoxy” refers to the alkoxygroups having about 1-7 preferably about 1-4 carbons.

The term “aryl” refers to monocyclic or bicyclic aromatic hydrocarbongroups having 6-20 carbon atoms in the ring portion. Preferably, thearyl is a (C₆-C₁₀)aryl. Non-limiting examples include phenyl, biphenyl,naphthyl or tetrahydronaphthyl, each of which may optionally besubstituted by 1-4 substituents, such as alkyl, trifluoromethyl,cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)—O—, aryl-O—,heteroaryl-O—, amino, HS—, alkyl-S—, aryl-S—, nitro, cyano, carboxy,alkyl-O—C(O)—, carbamoyl, alkyl-S(O)—, sulfonyl, sulfonamido,heterocyclyl and the like, wherein R is independently hydrogen, alkyl,aryl, heteroaryl, aryl-alkyl-, heteroaryl-alkyl- and the like.

Furthermore, the term “aryl” as used herein, refers to an aromaticsubstituent which can be a single aromatic ring, or multiple aromaticrings that are fused together, linked covalently, or linked to a commongroup such as a methylene or ethylene moiety. The common linking groupalso can be a carbonyl as in benzophenone or oxygen as in diphenyletheror nitrogen as in diphenylamine.

As used herein, the term “halogen” or “halo” refers to fluoro, chloro,bromo, and iodo.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thecompounds of this invention and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto. Pharmaceuticallyacceptable acid addition salts can be formed with inorganic acids andorganic acids. Inorganic acids from which salts can be derived include,for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The pharmaceutically acceptable salts of the presentinvention can be synthesized from a parent compound, a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are preferred, where practicable. Lists of additionalsuitable salts can be found, e.g., in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), whichis herein incorporated by reference.

In another embodiment the compound of Formula (I) is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogenphosphate salt.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The term “therapeutically effective amount” of a compound of the presentinvention refers to an amount of the compound of the present inventionthat will elicit the biological or medical response of a subject, orameliorate symptoms, slow or delay disease progression, or prevent adisease, etc. In one non-limiting embodiment, the term “atherapeutically effective amount” refers to the amount of the compoundof the present invention that, when administered to a subject, iseffective to

(1) at least partially alleviating, inhibiting, preventing and/orameliorating a condition, or a disorder or a disease (i) characterisedin excessive stress hormone levels and/or insufficient androgen hormonelevels or (ii) associated with activities of excessive stress hormonelevels and/or insufficient androgen hormone levels, or (iii)characterised by abnormal activities of excessive stress hormone levelsand/or insufficient androgen hormone levels; or(2) reducing or inhibiting the activities of excessive stress hormonelevels and/or reducing or inhibiting the activity of steroidogenicenzymes that indirectly lead to insufficient androgen hormone levels, or(3) reducing or inhibiting the synthesis of excessively produced stresshormone levels and/or increasing androgen hormone levels.

In another non-limiting embodiment, the term “a therapeuticallyeffective amount” refers to the amount of the compound of the presentinvention that, when administered to a cell, or a tissue, or anon-cellular biological material, or a medium, is effective to at leastpartially reducing or inhibiting the activities of excessive stresshormone levels and/or increasing androgen hormone levels; or at leastpartially reducing or inhibiting the synthesis of excessively producedstress hormone levels and/or increasing androgen hormone levels.

As used herein, the term “subject” refers to an animal. In oneembodiment, the animal is a mammal. A subject also refers to forexample, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice, fish, birds and the like. In one embodiment,the subject is a human.

As used throughout in this patent application, the levels of thehormones as measured in a subject could be in any sample taken from saidsubject. In one embodiment the levels are measured in a blood sample. Inanother embodiment the levels are determined from a plasma sample.

As used herein, the term “a disorder” or “a disease” refers to anyderangement or abnormality of function; a morbid physical or mentalstate. See Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co.27th ed. 1988).

As used herein, the term “treating” or “treatment” of any disease ordisorder refers in one embodiment, to partially or totally amelioratingthe disease or disorder (i.e., arresting or reducing the development ofthe disease or at least one of the clinical symptoms thereof). Inanother embodiment “treating” or “treatment” refers to partially ortotally ameliorating at least one physical parameter, which may not bediscernible by the patient. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers topreventing or delaying the onset or development or progression of thedisease or disorder.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Any asymmetric carbon atom on the compounds of the present invention canbe present in the (R)-, (S)- or (R,S)-configuration, preferably in the(R)- or (S)-configuration. Substituents at atoms with unsaturated bondsmay, if possible, be present in cis-(Z)- or trans (E)-form. Therefore,the compounds of the present invention can be in the form of one of thepossible isomers or mixtures thereof, for example, as substantially puregeometric (cis or trans) isomers, diastereomers, optical isomers(antipodes), racemates or mixtures thereof.

Any resulting mixtures of isomers can be separated on the basis of thephysicochemical differences of the constituents, into the pure geometricor optical isomers, diastereomers, racemates, for example, bychromatography and/or fractional crystallization.

Any resulting racemates of final products or intermediates can beresolved into the optical antipodes by known methods, e.g., byseparation of the diastereomeric salts thereof, obtained with anoptically active acid or base, and liberating the optically activeacidic or basic compound. In particular, the imidazolyl moiety may thusbe employed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, di-O, O′-p-toluoyl tartaric acid, mandelicacid, malic acid or camphor-10-sulfonic acid. Racemic products can alsobe resolved by chiral chromatography, e.g., high pressure liquidchromatography (HPLC) using a chiral adsorbent.

In addition, the present invention contemplates compounds of Formula (I)to include the free form, a salt form, or prodrug derivatives thereof.The compounds can be obtained in the form of hydrates or includesolvents used for their crystallization.

The compounds of the present invention can be synthesized or producedand characterized by methods as described in WO2007/024945, the contentsthereof is herein incorporated by reference.

In another embodiment, the methods include using the compounds accordingto Formula (I) to treat diseases or disorder described above, whereinthe compounds, including isomers, optical isomers or pharmaceuticallyacceptable salts thereof, preferably including isomers, optical isomers,are selected from

-   4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;-   3-bromo-4-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)benzonitrile;-   5-(2-chloro-4-cyanophenyl)-N-(4-methoxybenzyl)-N-methyl-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxamide;-   5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylic    acid (4-fluorobenzyl)methylamide;-   4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile;-   5-(3-fluoro-4-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole;-   5-(2-Chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylic    acid 4-fluorobenzyl ester;-   5-(2-Bromo-4-fluorophenyl)-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepine;-   2-Bromo-4-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)benzonitrile;-   3-Pyridin-3-yl-4-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)benzonitrile;    and-   3-Chloro-4-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)benzonitrile;    in particular selected from-   4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;-   3-bromo-4-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)benzonitrile;-   5-(2-chloro-4-cyanophenyl)-N-(4-methoxybenzyl)-N-methyl-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxamide;    and-   4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile.

Preferably a compound of formula (I), as described herein, is of theformula(4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitrileor a pharmaceutically acceptable salt thereof, in particular(4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate. A salt of a compound of formula (I), as definedherein, preferably a phosphate salt, such dihydrogen phosphate, may beprepared according to standard methods known to the person skilled inthe art, for example as described in Chem. Commun., 2007, 419-421(2007); in Development of a pharmaceutical cocrystal of a monophosphatesalt with phosphoric acid Alex M. Chen, Martha E. Ellison, AndreyPeresypkin, Robert M. Wenslow, Narayan Variankaval, Cecile G. Savarin,Theresa K. Natishan, a David J. Mathre, a Peter G. Dormer, a Danielle H.Euler, b Richard G. Ball, Zhixiong Ye, Yaling Wanga and Ivan Santos; inHandbook of Pharmaceutical Salts: Properties, Selection, and Use Editedby P. Heinrich Stahl and Camile G. Wermuth. VHCA, Verlag HelveticaChimica Acta, Zürich, Switzerland, and Wiley-VCH, Weinheim, Germany.2002; in Organic Process Research & Development 2000, 4, 427-435 SaltSelection and Optimisation Procedures for Pharmaceutical New ChemicalEntities Richard J. Bastin, Michael J. Bowker, and Bryan J. Slater; inAdvanced Drug Delivery Reviews 56 (2004) 275-300, High-throughputcrystallization: polymorphs, salts, co-crystals and solvates ofpharmaceutical solids Sherry L. Morissettea,*, O″rn Almarssona, MatthewL. Petersona, Julius F. Remenara, Michael J. Reada, Anthony V. Lemmoa,Steve Ellisa, Michael J. Cimab, Colin R. Gardner; and in Journal ofPharmaceutical Sciences, VOL. 96, NO. 5, MAY 2007, Structure,Solubility, Screening, and Synthesis of Molecular Salts, Black, S, N.,Collier, E. A., Davey, R. J. and Roberts, R. J.

According to the invention a compound of formula (I) and/or apharmaceutically acceptable salt thereof represents a pleiotropicmodifier of adrenal steroidogenesis when administered to a subject. Acompound of formula (I) maintains or lowers cortisol levels whenadministered to a subject. A compound of formula (I) increases11-deoxycortisol levels when administered to a subject. A compound offormula (I) increases levels of adreonocorticotropin when administeredto a subject. A compound of formula (I) increases levels of11-deoxycorticosterone. A compound of formula (I) increases adrenalandrogens when administered to a subject.

In example 1, it has been shown that a compound of formula (I) namely(4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate represents a pleiotropic modifier of adrenalsteroidogenesis when administered to subject. It is shown in FIG. 1,that4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate maintains or lowers cortisol levels whenadministered to a subject. It is further shown that4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate increases 11-deoxycortisol levels when administeredto a subject.4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate increases levels of adreonocorticotropin whenadministered to a subject.4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate increases levels of 11-deoxycorticosterone.4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate increases adrenal androgens when administered to asubject.

The clinical relevance of increased stress hormone levels and/ordecreased androgen hormone levels has been shown for the followingconditions.

(i) chronic heart failure(ii) chronic heart failure with impaired exercise tolerance(iii) chronic heart failure with muscle weakness(iv) cardiac cachexia(v) COPD-induced cachexia(vi) cirrhosis-induced cachexia(vii) tumor-induced cachexia(viii) viral (HIV)-induced cachexia(ix) acute heart failure (x) acute decompensated heart failure(xi) acute coronary syndrome(xii) chronic stress syndrome(xiii) Cushing's syndrome(xiv) metabolic syndrome(xv) hypercortisolemia

(i) Chronic heart failure as well as chronic heart failure conditionswith impaired exercise tolerance (ii) and muscle weakness (iv) showraised plasma aldosterone levels as shown by Bolger et al. Circulation2002; 106:92-99, a raised plasma to dihydroepiandrosterone ratio asshown by Anker et al. European Heart Journal 1999; 20:683-693 anddecreased androgen levels as shown by Jankowaska et al., Circulation2006; 114:1829-1837.

(iv) Cardiac cachexia is a serious complication of chronic heart failureas patients suffer from a general loss of fat tissue, lean tissue andbone tissue. Cardiac cachexia patients show raised plasma levels ofaldosterone and cortisol as well as reduced levels ofdehydroepiandrosterone as described by Anker et al., Circulation 1997;96:526-534 and illustrated in WO 2000/21509 and US 2009/0023639.

(v) COPD-induced cachexia, cirrhosis-induced cachexia (vi),tumor-induced cachexia (vii) and viral (HIV)-induced cachexia (viii) arecharacterized by increased plasma aldosterone levels as documented in WO2000/21509 or US 2009/0023639 and have been treated with anabolicandrogen or androgen-derivatives as reported by Yeh et al., Chest 2002;122:421-428 and by Cuerda et al., Nutrition Clinical Practice 2005 20;93-97.

(ix-x) Plasma cortisol predict cardiac events such as death andhospitalization in patients with heart failure according to Yamaji etal. Circulation Heart Failure 2009; 2:608-613.

(xi) Myocardial infarction raises cortisol levels that affect cardiacremodeling as indicated by Mihailidu et al., Hypertension 2009 in press.The magnitude of the cortisol response is related to the size of theensuing infarction as shown by Bain et al., International Journal ofCardiology 1992; 27:145-150.

(xii) Chronic stress disorders with its physical and psychologicalramifications has been associated with excessive aldosterone andcortisol levels according to Kubzansky and Adler, Neuroscience andBiobehavioral Reviews, 2009; 5:1-7. Particularly, excessive andpersistent cortisol secretion can lead to depression, hyperglycemia andthe suppression of the immune system.

(xiii) Cushing's syndrome describes a condition of chronically excessivecortisol release. The cortisol excess may orginate directly from anadrenocortical tumor or secondarily from a pituitary (Cushing's Disease)or ectopic tumor that releases adrenocorticotropin as illustrated byBoscaro and Arnaldi, Journal of Clinical Endocrinology and Metabolism2009; 94:3121-3131.

(xiv) Metabolic syndrome defines a state of metabolic dysregulationcharacterized by insulin resistance and a predisposition to type 2diabetes, central and visceral obesity, hypertension and dyslipidemia.The metabolic dysregulation can be caused by an underlying endocrineimbalance mediated by the adrenal steroids aldosterone and cortisol asreported by Kidamby et al. Hypertension 2007; 49:704-711.

(xv) Hypercortisolemia refers to conditions that are characterized byhigh levels of circulating cortisol. High levels of plasma cortisol maydirectly contribute to a pathological condition, represent a sign of apathological condition or be of non-pathological nature.

The invention relates to the use of a compound of formula (I) and/or apharmaceutically acceptable salt thereof or any other form of it as asdiscussed above with adrenal hormone-modifying properties as treatmentfor conditions that are characterized by excessive stress hormone levelsand or insufficient androgen hormone levels such as heart failure,cachexia, acute coronary syndrome, chronic stress syndrome,hypercortisolemia, cushing's syndrome or metabolic syndrome, inparticular heart failure, cachexia, acute coronary syndrome, chronicstress syndrome, cushing's syndrome or metabolic syndrome. Heart failurecould be both acute heart failure and chronic heart failure. Acute heartfailure could be acute decompensated heart failure. Chronic heartfailure could be associated with impaired exercise tolerance and or withmuscle weakness. Cachexia could be cardiac cachexia, COPD-inducedcachexia, cirrhosis-induced cachexia tumor-induced cachexia or viral(HIV)-induced cachexia. Chronic stress syndrome may include depression,hyperglycemia and immunesuppression. Cushing's syndrome can includehypercortisolism due to adrenocortical, pituitary or ectopic tumors.Metabolic syndrome could include obesity, diabetes, hypertension,dyslipidemia and atherosclerosis. The compounds of Formula (I) haveadrenal hormone-modifying properties in diseases or conditionscharacterised by increased stress hormone levels and/or decreasedandrogen hormone levels as shown in the experimental section.

The present invention provides a method of lowering or maintainingcortisol levels in a subject by administering a therapeuticallyefficient dose of a compound of formula (I).

The present invention provides a method of treating a disorder, diseaseor condition characterised by decreased or insufficient androgen hormonelevels in a subject by administering a therapeutically efficient dose ofa compound of formula (I).

The present invention provides a compound of formula (I), orpharmaceutically acceptable salt thereof, for use in the treatment of adisorder, disease or condition characterised by decreased orinsufficient androgen hormone levels.

The present invention provides a method of treating a disorder, diseaseor condition characterised by excessive stress hormone levels, such asaldesterone and cortisol levels in a subject by administering atherapeutically efficient dose of a compound of formula (I).

The present invention provides a compound of formula (I), orpharmaceutically acceptable salt thereof, for use in the treatment of adisorder, disease or condition characterised by excessive stress hormonelevels, such as aldesterone and cortisol levels

The present invention provides a method of increasing or maintaining11-deoxycortisol levels levels in a subject by administering atherapeutically efficient dose of a compound of formula (I).

The present invention provides a method of increasing or maintainingadreonocorticotropin levels levels in a subject by administering atherapeutically efficient dose of a compound of formula (I).

The present invention provides a method of increasing or maintaining11-deoxycorticosterone levels in a subject by administering atherapeutically efficient dose of a compound of formula (I).

The present invention provides a method of increasing or maintainingadrenal androgens levels levels in a subject by administering atherapeutically efficient dose of a compound of formula (I).

The present invention provides the use of a compound of Formula (I) aspleiotropic modifier of adrenal steroidogenesis in a subject.

It further provides the use of a compound according to Formula (I) forthe preparation of a pharmaceutical composition for the treatment of adisorder, disease or condition characterised by excessive stress hormonelevels and/or insufficient androgen hormone levels.

It further provides a pharmaceutical composition comprising a compoundaccording to formula (I) for use in the treatment of a disorder, diseaseor condition characterised by excessive stress hormone levels and/orinsufficient androgen hormone levels. Such diseases or disorders can beheart failure, cachexia, acute coronary syndrome, chronic stresssyndrome, hypercortisolemia, cushing's syndrome or metabolic syndrome.

It further provides the use of a compound of the present invention forthe preparation of a pharmaceutical composition for the treatment of adisorder or disease or condition characterised by excessive stresshormone levels and or insufficient androgen hormone levels such as suchas heart failure, cachexia, acute coronary syndrome, chronic stresssyndrome, cushing's syndrome or metabolic syndrome.

In another aspect, the present invention provides methods of treatingdiseases or disorders characterised by excessive stress hormone levelsand or insufficient androgen hormone levels by administering to asubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound according to Formula (I) and apharmaceutically acceptable carrier. In another aspect, such diseases ordisorders can be heart failure, cachexia, acute coronary syndrome,chronic stress syndrome, cushing's syndrome or metabolic syndrome.

In one embodiment, the present invention provides methods ofadministering to a subject a therapeutically effective amount of apharmaceutical composition comprising a compound according to Formula(I) and a pharmaceutically acceptable carrier, to treat diseases ordisorders characterised by excessive stress hormone levels and orinsufficient androgen hormone levels. In another aspect, such diseasesor disorders can be heart failure, cachexia, acute coronary syndrome,chronic stress syndrome, hypercortisolemia, cushing's syndrome ormetabolic syndrome; in particular heart failure, cachexia, acutecoronary syndrome, chronic stress syndrome, cushing's syndrome ormetabolic syndrome.

Whenever used above, heart failure could be both acute heart failure andchronic heart failure. Acute heart failure could be acute decompensatedheart failure. Chronic heart failure could be associated with impairedexercise tolerance and or with muscle weakness. Cachexia could becardiac cachexia, COPD-induced cachexia, cirrhosis-induced cachexiatumor-induced cachexia or viral (HIV)-induced cachexia. Chronic stresssyndrome may include depression, hyperglycemia and immunesuppression.Cushing's syndrome can include hypercortisolism due to adrenocortical,pituitary or ectopic tumors. Metabolic syndrome could include obesity,diabetes, hypertension, dyslipidemia and atherosclerosis. The compoundsof Formula (I) have adrenal hormone-modifying properties in diseases orconditions characterised by increased stress hormone levels and/ordecreased androgen hormone levels as shown in the experimental section.

A pharmaceutical composition comprising a compound of the presentinvention may be prepared according to methods known in the art. Thepharmaceutical composition can be formulated for particular routes ofadministration such as oral administration, parenteral administration,and rectal administration, etc. In addition, the pharmaceuticalcompositions of the present invention can be made up in a solid formincluding capsules, tablets, pills, granules, powders or suppositories,or in a liquid form including solutions, suspensions or emulsions. Thepharmaceutical compositions can be subjected to conventionalpharmaceutical operations such as sterilization and/or can containconventional inert diluents, lubricating agents, or buffering agents, aswell as adjuvants, such as preservatives, stabilizers, wetting agents,emulsifers and buffers etc.

The pharmaceutical composition according to the present invention can bein unit dosage of at least 0.05 or 1 mg or greater of the compoundsdescribed herein as the active ingredient, such as of from 0.01 mg to1000 mg, of from 0.01 mg to 500 mg, of from 0.01 to 50 mg, of from 0.01mg to 5 mg, of from 0.01 to 2 mg or of from 0.1 mg to 2 mg of activeingredient; such as in unit dosage of at least 0.05 or 1 mg or of from 4mg to 100 mg, for example of from 2 mg to 50 mg, of the compoundsdescribed herein as the active ingredient for a subject of about 50-70kg. For example, the unit dosage can contain 1-1000 mg of activeingredient for a subject of about 50-70 kg, about 1-500 mg, about 1-50mg, about 0.5-5 mg, 0.1-1 mg or about 0.05-0.5 mg of active ingredient.The dosage regimen utilizing the compounds described herein can beselected in accordance with a variety of factors including type,species, age, weight, sex, the type of disease or disorder to betreated, the severity of the disease or disorder to be treated, theroute of administration, and the particular compound or salt employed. Aphysician, clinician or veterinarian of ordinary skill can readilydetermine the effective amount of each of the active ingredientsnecessary to prevent, treat or inhibit the progress of the disorder ordisease.

The above-cited dosage properties are demonstrable in vitro (See PCTapplications PCT/US2007/018660 & WO2007/065942A2) and in vivo tests (SeeExample 1 below) using advantageously mammals, e.g., mice, rats, dogs,monkeys or isolated organs, tissues and preparations thereof. Thecompounds of the present invention can be applied in vitro in the formof solutions, e.g., preferably aqueous solutions, and in vivo eitherenterally, parenterally, advantageously intravenously, e.g., as asuspension or in aqueous solution. The dosage in vitro may range betweenabout 10⁻³ molar and 10⁻⁹ molar concentrations. A therapeuticallyeffective amount in vivo may range depending on the route ofadministration, between 0.001-15 mg/kg, preferably between 0.003-0.05mg/kg.

Listed below are definitions of various terms used throughout thespecification:

The term “stress hormone”, as used herein, relates to a hormone which issecreted in response to an unusual exposure to life. The stress responseinvolves the activation of both the sympathetic adrenomedullary systemwith the secretion of epinephrine and norepinephrine, and thehypothalamic pituitary adrenocortical (HPA) system with the secretion ofcortisol. Examples of stress hormones are, for example described inTable 1 in WO2007/105203, which is incorporated herein by reference. Ina preferred embodiment, a stress hormone is aldosterone or cortisol,preferably cortisol.

The term “increased stress hormone levels” or “excessive stress hormonelevels” is used herein to indicate that the level of hormone levels isstatistically significantly raised relative to that of other parameters,such as aldosterone to plasma renin activity, or is statisticallysignificantly raised relative to normal clinical reference values. Forexample, stress hormone levels are increased if the level of aldosteroneis above 277 μM at rest or if the level of cortisol is above 552 nM inthe morning, as described, for example in Endocrinology 9^(th) eidtions(Editors J. D. Wilson, D. W. Foster, H. M. Kronenberg, P. R. Larsen) W.B. Saunders Co., Philadelphia, 1988.

The term “reducing or inhibiting the activities of excessive stresshormone levels”, as used herein, means any improvement in preventing,controlling, delaying, abating or mitigating relative and/or absoluteincreased or excessive stress hormone dysbalances leading topathophysiology. Although the term “inhibiting” is not intended to berestricted to the normalization of stress hormone levels, it alsoincludes the possibility that stress hormone levels are entirelynormalized to clinical reference values.

The term “reducing or inhibiting the synthesis excessively producedstress hormone levels”, as used herein, means any improvement inpreventing, controlling, delaying, abating or mitigating relative and/orabsolute increased or excessive stress hormone dysbalances leading topathophysiology. Although the term “inhibiting” is not intended to berestricted to the normalization of stress hormone levels, it alsoincludes the possibility that stress hormone levels are entirelynormalized to clinical reference values.

The term “androgen hormone”, as used herein, relates to male sexhormones and includes, for example, dehydroepiandrosterone sulphate(DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A),testosterone (T) and dihydrotestosterone (DHT).

The terms “decreased androgen hormone levels” or “insufficient androgenhormone levels” are used herein to indicate that the level of androgenlevels is statistically significantly lowered relative to that of otherparameters, or is statistically significantly lowered relative to normalclinical reference values. For example, androgen hormone levels aredecreased or insufficient if the level of androstendione is, forexample, below 2619 μM or if the level of dehydroandrosterone is, forexample, below 6.94 nM, as described, for example in Endocrinology9^(th) editions (Editors J. D. Wilson, D. W. Foster, H. M. Kronenberg,P. R. Larsen) W. B. Saunders Co., Philadelphia, 1988.

The term “reducing or inhibiting the activity of steroidogenic enzymesthat indirectly lead to insufficient androgen hormone levels”, as usedherein, means any improvement in preventing, controlling, delaying,abating or mitigating relative and/or absolute decreased or insufficientandrogen hormone dysbalances leading to pathophysiology. Although theterm “inhibiting” is not intended to be restricted to the normalizationof androgen hormone levels, it also includes the possibility thatandrogen hormone levels are entirely normalized to clinical referencevalues.

The term “maintain” or “maintaining”, when referring to hormone levelsis used herein to mean an improvement in preventing, controlling ordelaying relative and/or absolute decreased/insufficient androgenhormone levels and/or relative and/or absolute increased/excessivestress hormone levels.

The term “lower” or “lowering”, when referring to stress hormone levelsis used herein to mean any improvement in abating relative and/orabsolute increased/excessive stress hormone levels.

The term “increase” or “increasing”, when referring to androgen hormonelevels is used herein to mean any improvement in mitigating relativeand/or absolute decreased/insufficient androgen hormone levels.

As used herein, the term “abnormal” refers to an activity or featurewhich differs from a normal activity or feature.

The term “abnormal activity”, as used herein, refers to any derangementof normal function. The abnormal activity can be stronger or weaker thanthe normal activity. In one embodiment, the “abnormal activity” refersto either over- or under-activity of, for example, and hormone, asdefined herein.

The term “activity”, as used herein, refers to any specific activitythat a molecule is capable of performing or encoding. For exampleactivity may be that a molecule is capable of associating with aspecific binding partner with a specific affinity, capable of catalyzinga specific reaction, capable of inhibiting a specific reaction orcapable of effecting a particular cellular response.

The term “expression”, as used herein, is to be understood as defined,for example in Maniatis et al “Molecular Cloning: A Laboratory Manual”Cold Spring Harbor Laboratory Press: 2^(nd) Edition, 1989, for example,it refers to the accumulation of a molecule, such as an hormone asdefined herein.

The term “pleiotropic adrenal hormone-modifying agent”, as used herein,is to be understood as a molecule, such as a compound of formula (I) asdefined herein, which inhibits the synthesis of both, aldosterone andcortisol, while it increases the levels of ACTH, 11-deoxycorticosteroneand the synthesis of the adrenal androgens, androstendione anddehydroepiandrosterone.

The term “substituted”, as used herein, refers to one or moresubstituents, for example one or two substituents, for examplesubstituents as defined herein for a compound of formula (I).

The term “Cushing's syndrome” is also referred to ashyperadrenocorticism or hypercorticism. Cushing's syndrome can includehypercortisolism due to adrenocortical, pituitary or ectopic tumors.

For the purpose of this invention, the compound of formula (I), asdefined herein, refers to both the free form as well as anypharmaceutical acceptable salt thereof.

EXPERIMENTAL SECTION

The following examples illustrate the above-described invention.However, it is not intended to restrict the scope of this invention inany manner. Other embodiments will be evident to a person skilled in theart whilst reading the foregoing detailed description The scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

Example 1 In Vitro Rat CYP11B1 Assay

Complete EDTA-free protease inhibitor tablets were obtained from RocheApplied Science (Indianapolis, Ind.). Dulbecco's modified Eagle medium(DMEM), antibiotic, geneticin, hygromycin, and fetal bovine serum (FBS)were products of Invitrogen (Carlsbad, Calif.). NADPH RegenerationSolution A and Solution B were purchased from BD Biosciences Clontech(Palo Alto, Calif.). Anti-sheep PVT SPA beads and[1,2,6,7-³H(N)]corticosterone were acquired from Amersham (Piscataway,N.J.) and PerkinElmer (Boston, Mass.), respectively.

Cell line V79-4 CYP11B1-adrenodoxin-adrenodoxin reductase #259 wasmaintained in DMEM supplemented with 10% FBS, 0.5× antibiotic, 800 μg/mlgeneticin, and 250 μg/ml hygromycin (double-selection medium). Forenzyme preparation, #259 cells were seeded in 150 mm dishes indouble-selection medium. Following 2 days of growth, cells were washedonce with PBS, scraped and collected in PBS, and centrifuged at 1,300rpm for 6 min. Each pellet (representing 10 dishes of cells) wasresuspended in 3 ml of ice-cold homogenization buffer (8.5 mM MgCl₂,3.13 mM KCl, 7.59 mM NaCl, 50 mM Tris/HCl, pH 7.4, and one completeEDTA-free protease inhibitor tablet per 100 ml buffer), sonicated usinga Branson Sonifier 450 with 6 pulses, and then placed on ice for 5 min.The sonication procedure was repeated 3 more times, with a 5 min rest onice between sonications. The sonicated material was then spun at 500×gfor 4 min to remove unbroken cells. The supernatant was brought to afinal glycerol concentration of 5%, flash-frozen in liquid nitrogen, andstored at −80° C.

Material from frozen CYP11B1 preparations was thawed on ice on the dayof experiment and then diluted in an ice-cold assay buffer containing8.5 mM MgCl₂, 3.13 mM KCl, 7.59 mM NaCl, and 50 mM Tris/HCl, pH 7.4, toa protein concentration of 0.5-6 mg/ml. The CYP11B1 assays wereperformed in 96-well U-bottom non-tissue-culture-treated plates.Depending on the experiment, 50 to 300 μg of protein in 35 μl wasincubated with 75 μl of assay buffer or a compound at the desiredconcentration and 20 μl of substrate mix (1.08×NADPH RegenerationSolution A, 6.5×NADPH Regeneration Solution B, 811 μM NADPH, and 3.25 μM11-deoxycorticosterone in assay buffer) for up to 4 hr at 25° C. in ashaking incubator. The reaction was stopped by adding 10 μl of 1.4%Triton X-100 and briefly shaking the plates. Plates were thencentrifuged at 2,400 rpm for 6 min, and 50 μl of supernatant was removedfor measurement of corticosterone content by scintillation proximityassay (SPA).

Measurement of corticosterone was performed using a 96-well plateformat. Each test sample (50 μl) was incubated with 0.02 μCi of[1,2,6,7-³H(N)]corticosterone and 0.3 μg of anti-corticosterone antibodyin PBS containing 0.1% Triton X-100, 0.1% bovine serum albumin, and 12%glycerol in a total volume of 200 μl at room temperature for 1 hr.Anti-sheep PVT SPA beads (50 μl) were then added to each well andincubated overnight at room temperature prior to counting in a Microbetaplate counter. The amount of corticosterone in each sample wascalculated by comparing with a standard curve generated using knownquantities of the hormone.

Full concentration-response curves of an inhibitor were performed atleast 3 times. The IC₅₀ values were derived using a non-linear leastsquares curve-fitting program from IDBS XLfit. It has been found thatthe compounds within the scope of the present invention, particularlythe specific compounds disclosed herein are active CYP11B1 inhibitorshaving IC₅₀ ranging from 0.3 nM to 600 nM.

Example 2 In Vitro Rat CYP11B2 Assay

Cell line V79-4 rCYP11B2-adrenodoxin-adrenodoxin reductase #305 ismaintained in DMEM supplemented with 10% FBS, 0.5× antibiotic, 800 μg/mlgeneticin, and 250 μg/ml hygromycin (double-selection medium). Forenzyme preparation, #305 cells are seeded in 150 mm dishes (doubleselection medium) with an approximate surface area split of 1:15 fromT-185 flask cultures growing at 75-85% confluence. Following 2 days ofgrowth, cells are washed once with PBS, scraped and collected in PBS,and centrifuged at 1,300 rpm for 6 min. Each pellet (representing 10dishes of cells) is resuspended in 3 ml of ice-cold homogenizationbuffer (8.5 mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, 50 mM Tris/HCl, pH 7.4,and one complete EDTA-free protease inhibitor tablet per 100 ml buffer),sonicated using a Branson Sonifier 450 with 6 pulses, and then placed onice for 5 min. The sonication procedure is repeated 3 more times, with a5 min incubation on ice between sonications. The sonicated material isthen spun at 500×g for 4 min to remove unbroken cells. The supernatantis brought to a final glycerol concentration of 5%, flash-frozen inliquid nitrogen, and stored at −80° C.

Material from frozen CYP11B2 preparations is thawed on ice on the day ofexperiment and then diluted in an ice-cold assay buffer containing 8.5mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, and 50 mM Tris/HCl, pH 7.4, to aprotein concentration of 0.25-1.5 mg/ml. The CYP11B2 assay is performedin 96-well U-bottom non-tissue-culture-treated plates. Depending on theexperiment, 14 to 84 μg of protein in 55 μl is incubated with 75 μl ofassay buffer or a compound at the desired concentration and 20 μl ofsubstrate mix (1.25×NADPH Regeneration Solution A, 7.5×NADPHRegeneration Solution B, 935.75 μM NADPH, and 15 μM11-deoxycorticosterone in assay buffer) for up to 5 hr at 25° C. in ashaking incubator. The reaction is stopped by adding 10 μl of 1.6%Triton X-100 and briefly shaking the plates. Plates are then centrifugedat 2,400 rpm for 6 min, and 100 μl of supernatant is removed formeasurement of aldosterone content by scintillation proximity assay(SPA).

Measurement of aldosterone is performed using a 96-well plate format.Each test sample (2-10 μl of cell culture medium or 100 μl of cellhomogenate) is incubated with 0.02 μCi of [1,2,6,7-3H(N)]aldosterone and0.3 μg of anti-aldosterone antibody in PBS containing 0.1% Triton X-100,0.1% bovine serum albumin, and 12% glycerol in a total volume of 200 μlat room temperature for 1 hr. Anti-mouse PVT SPA beads (50 μl) are thenadded to each well and incubated for 4 hr at room temperature prior tocounting in a Microbeta plate counter. The amount of aldosterone in eachsample is calculated by comparing with a standard curve generated usingknown quantities of the hormone.

Full concentration-response curves of an inhibitor are performed atleast 3 times. The IC50 values are derived using a non-linear leastsquares curve-fitting program from DBS XLfit.

Example 3 In Vitro Human CYP11B1 Assay

Complete EDTA-free protease inhibitor tablets were obtained from RocheApplied Science (Indianapolis, Ind.). Dulbecco's modified Eagle medium(DMEM), antibiotic, geneticin, hygromycin, and fetal bovine serum (FBS)were products of Invitrogen (Carlsbad, Calif.). NADPH RegenerationSolution A and Solution B were purchased from BD Biosciences Clontech(Palo Alto, Calif.). Anti-mouse PVT SPA beads and[1,2,6,7-³H(N)]hydrocortisone were acquired from Amersham (Piscataway,N.J.) and PerkinElmer (Boston, Mass.), respectively.

Cell line V79-4 CYP11B1-adrenodoxin-adrenodoxin reductase #618 wasmaintained in DMEM supplemented with 10% FBS, 0.5× antibiotic, 800 μg/mlgeneticin, and 250 μg/ml hygromycin (double-selection medium). Forenzyme preparation, #618 cells were seeded in 150 mm dishes at 6.75×10⁵cells per dish in double-selection medium. Following 4 days of growth,cells were washed once with PBS, scraped and collected in PBS, andcentrifuged at 1,300 rpm for 6 min. Each pellet (representing 10 dishesof cells) was resuspended in 3 ml of ice-cold homogenization buffer (8.5mM MgCl₂, 3.13 mM KCl, 7.59 mM NaCl, 50 mM Tris/HCl, pH 7.4, and onecomplete EDTA-free protease inhibitor tablet per 100 ml buffer),sonicated using a Branson Sonifier 450 with 6 pulses, and then placed onice for 5 min. The sonication procedure was repeated 3 more times, witha 5 min rest on ice between sonications. The sonicated material was thenspun at 500×g for 4 min to remove unbroken cells. The supernatant wasbrought to a final glycerol concentration of 5%, flash-frozen in liquidnitrogen, and stored at −80° C.

Material from frozen CYP11B1 preparations was thawed on ice on the dayof experiment and then diluted in an ice-cold assay buffer containing8.5 mM MgCl₂, 3.13 mM KCl, 7.59 mM NaCl, and 50 mM Tris/HCl, pH 7.4, toa protein concentration of 0.5-6 mg/ml. The CYP11B1 assays wereperformed in 96-well U-bottom non-tissue-culture-treated plates.Depending on the experiment, 50 to 300 μg of protein in 35 μl wasincubated with 75 μl of assay buffer or a compound at the desiredconcentration and 20 μl of substrate mix (1.08×NADPH RegenerationSolution A, 6.5×NADPH Regeneration Solution B, 811 μM NADPH, and 3.25 μM11-deoxycortisol in assay buffer) for up to 4 hr at 25° C. in a shakingincubator. The reaction was stopped by adding 10 μl of 1.4% Triton X-100and briefly shaking the plates. Plates were then centrifuged at 2,400rpm for 6 min, and 50 μl of supernatant was removed for measurement ofcortisol content by scintillation proximity assay (SPA). Measurement ofcortisol was performed using a 96-well plate format. Each test sample(50 μl) was incubated with 0.02 μCi of [1,2,6,7-³H(N)]hydrocortisone and0.3 μg of anti-cortisol antibody in PBS containing 0.1% Triton X-100,0.1% bovine serum albumin, and 12% glycerol in a total volume of 200 μlat room temperature for 1 hr. Anti-mouse PVT SPA beads (50 μl) were thenadded to each well and incubated overnight at room temperature prior tocounting in a Microbeta plate counter. The amount of cortisol in eachsample was calculated by comparing with a standard curve generated usingknown quantities of the hormone. Full concentration-response curves ofan inhibitor were performed at least 3 times. The IC₅₀ values werederived using a non-linear least squares curve-fitting program from IDBSXLfit. It has been found that the compounds within the scope of thepresent invention, particularly the specific compounds disclosed hereinare active CYP11B1 inhibitors having IC₅₀ ranging from 0.2 nM to 200 nM.

Example 4 In Vitro Human CYP11B2 (aldesterone) Assay

Human adrenocortical carcinoma NCI-H295R cell line was obtained fromAmerican Type Culture Collection (Manassas, Va.).Insulin/transferrin/selenium (ITS) —A supplement (100×), DMEM/F-12,antibiotic/antimycotic (100×), and fetal bovine serum (FBS) werepurchased from Invitrogen (Carlsbad, Calif.). Anti-mouse PVTscintillation proximity assay (SPA) beads and NBS 96-well plates wereobtained from GE Health Sciences (Piscataway, N.J.) and Corning (Acton,Mass.), respectively. Solid black 96-well flat bottom plates werepurchased from Costar (Corning, N.Y.). Aldosterone and angiotensin (AngII) were purchased from Sigma (St. Louis, Mo.).D-[1,2,6,7-³H(N)]aldosterone was acquired from PerkinElmer (Boston,Mass.). Nu-serum was a product of BD Biosciences (Franklin Lakes, N.J.).

For in vitro measurement of aldosterone activity, human adrenocorticalcarcinoma NCl-H295R cells are seeded in NBS 96-well plates at a densityof 25,000 cells/well in 100 μl of a growth medium containing DMEM/F12supplemented with 10% FCS, 2.5% Nu-serum, 1 μg ITS/ml, and 1×antibiotic/antimycotic. The medium is changed after culturing for 3 daysat 37° C. under an atmosphere of 5% CO₂/95% air. On the following day,cells are rinsed with 100 μl of phosphate-buffered saline (PBS) andincubated with 100 μl of treatment medium containing 1 μM Ang II and acompound at different concentrations in quadruplicate wells at 37° C.for 24 hr. At the end of incubation, 50 μl of medium is withdrawn fromeach well for measurement of aldosterone production by an SPA usingmouse anti-aldosterone monoclonal antibodies.

Measurement of aldosterone activity can also be performed using a96-well plate format. Each test sample is incubated with 0.02 μCi ofD-[1,2,6,7-³H(N)]aldosterone and 0.3 μg of anti-aldosterone antibody inPBS containing 0.1% Triton X-100, 0.1% bovine serum albumin, and 12%glycerol in a total volume of 200 μl at room temperature for 1 hr.Anti-mouse PVT SPA beads (50 μl) are then added to each well andincubated overnight at room temperature prior to counting in a Microbetaplate counter. The amount of aldosterone in each sample is calculated bycomparing with a standard curve generated using known quantities of thehormone.

Example 5 Determination of IC₅₀ Values for CYP11B1 and CYP11B2

The excretion of aldosterone, cortisol, corticosterone andestradiol/estrone into the culture medium can be detected and quantifiedby commercially available, specific monoclonal antibodies inradioimmunoassays in accordance with the manufacturer's instructions.Inhibition of the release of certain steroids can be used as a measureof the respective enzyme inhibition by the added test compounds. Thedose-dependent inhibition of enzymic activity by a compound iscalculated by means of an inhibition plot which is characterized by anIC50. The IC50 values for active test compounds are ascertained by asimple linear regression analysis in order to construct inhibition plotswithout data weighting. The inhibition plot is calculated by fitting a4-parameter logistic function to the raw data points using the leastsquares method. The equation of the 4-parameter logistic function iscalculated as follows: Y=(d−a)/((1+(x/c)b))+a, where: a=minimum datalevel, b=gradient, I c=ICED, d=maximum data level, x=inhibitorconcentration.

The inhibition activity of aldosterone production can also be expressedin percentage inhibition (% inhibition) at a given concentration (e.g. %inhibition at 1 μM), which is the aldosterone level when the cell istreated with the given concentration of a compound of this invention(e.g. concentration of 1 μM) versus the aldosterone excretion when cellis free of the compound of the invention:

% inhibition aldosterone production=[(Y−X)/Y]×100

wherein X is the level of aldosterone when the cell is treated with acompound according to anyone of Formulae I to IVB; or pharmaceuticallyacceptable salt thereof, and Y is the level of aldosterone when the cellis free of compound according to anyone of Formulae I to IVB, orpharmaceutically acceptable salt thereof.

The inhibition activity of CYP11B1 production can also be expressed inpercentage inhibition (% inhibition) at a given concentration (e.g. %inhibition at 1 μM), which is the cortisol level when cell is treatedwith the given concentration of a compound of the invention (e.g.concentration of 1 μM) versus the cortisol excretion when cell is freeof the compound of the invention.

% inhibition cortisol production=[(Y′−X′)/Y′]×100

wherein X′ is the level of cortisol when the cell is treated with acompound of Formulae I to IVB; and Y′ is the level of cortisol when thecell is free of compound of Formulae I to IVB. Using the test assays formeasuring CYP11B1 (cortisol) and CYP11B2 (aldosterone), as describedabove, compounds of the invention exhibited inhibitory efficacy as shownin Table 1.

TABLE 1 Data from Example 1, 2, 3 and 4 Human Human Rat Rat CYP11B1CYP11B2 CYP11B1 CYP11B2 Compound nM nM nM nM 4′-fluoro-6-(6,7,8,9- 0.30.4 0.3 1.0 tetrahydro-5H- imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile enantiomer A** (Example 25 as described inWO2007/024945) 3-bromo-4-(6,7,8,9- 0.2 1.2 1.0 1.0 tetrahydro-5H-imidazo[1,5-a]azepin-5- yl)benzonitrile racemate** (Example 23 asdescribed in WO2007/024945) 5-(2-chloro-4- 0.8 2.7 2.0 5.0cyanophenyl)-N-(4- methoxybenzyl)-N- methyl-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole- 5-carboxamide entantiomer B** (Example 9-9 asdescribed in WO2007/024945) 4-(6,7-Dihydro-5H- 1.8 0.7 495 110pyrrolo[1,2-c]imidazol-5- yl)-3-fluorobenzonitrile enantiomer B** ((R)enantiomer, as described in Exampe 3, in WO2007/024945) **Please referto WO2007/024945 for details.

Example 6 In Vivo CYP11B1 Assay

The in vivo effects of compounds on plasma aldosterone concentration(PAC) and plasma glucocorticoid (corticosterone) concentration (PCC)were evaluated in conscious rats.

Male Sprague-Dawley rats (˜400-600 g body weight) were surgicallyinstrumented with a femoral arterial and venous catheter. The catheterswere exteriorized from the lower back through a stainless steel springand swivel system that enabled the rats to move freely at all times.Rats were allowed at least one week to recover from the surgery beforeinitiating the experiments.

On the morning of the experiment, a pretreatment blood sample wascollected on heparin from the arterial catheter. The blood samples werespun in a refrigerated centrifuge to generate plasma. The plasma wasstored frozen at −70° C. until the later measurement of PAC and PCC (byradioimmunoassay). Adrenocorticotropic hormone (ACTH(1-24), referred toherein as ACTH) was then administered as an intravenous (i.v.) bolus(100 ng/kg) followed by a continuous i.v. infusion (30 ng/kg/min) for 9hours. After one hour of infusion, a baseline (time 0) blood sample waswithdrawn from the arterial catheter and processed and stored asdescribed above. The rats were then dosed (typically 0.01 to 100 mg/kg)with the test compound p.o. by oral gavage or parenterally via thearterial catheter (i.a.). Compounds were formulated in an appropriatevehicle (e.g., water (p.o.) or saline (i.a.)) at a physiologicallycompatible volume (typically 1-2 ml/kg). Additional blood samples werewithdrawn at 0.083 (i.a. only), 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and24 hours after dosing with compound and processed and stored as abovefor later determination of PAC, PCC, and plasma compound concentration(by LC/MS/MS). Oral bioavailability and traditional pharmacokinetic (PK)parameters were estimated from the plasma compound concentrations.

In the control rats, ACTH administration resulted in a sustainedincrease in PAC by ˜10-fold (from ˜0.26 nM to ˜2.5 nM) and PCC by ˜4- to5-fold (from ˜300 nM to ˜1340 nM) for the duration of the 9-hourexperiment. In contrast, administration of a test compound time- anddose-dependently lowered PAC and PCC by 0 to 97% depending on thecompound's inherent CYP11B2 and CYP11B1 inhibitory potencies and itsADME (absorption, distribution, metabolism, excretion) properties. Basedon the plasma compound concentration at each dose, the PK/PD(pharmacodynamic) profiles (reduction of PAC and PCC) of each compoundwere determined. Table 2 below summarizes the CYP11B1 and CYP11B2inhibitory activities of representative compounds.

TABLE 2 % Reduction Dose from baseline Compound (mg/kg) t = 1 h 8 h4′-fluoro-6-(6,7,8,9-tetrahydro- 3 (i.a.) PCC 89 435H-imidazo[1,5-a]azepin-5- PAC 83 76 yl)biphenyl-3-carbonitrileenantiomer A** (Example 25 as described in WO2007/024945)3-bromo-4-(6,7,8,9-tetrahydro- 10 (p.o.) PCC 70 875H-imidazo[1,5-a]azepin-5- PAC 79 89 yl)benzonitrile racemate** (Example23 as described in WO2007/024945) 5-(2-chloro-4-cyanophenyl)-N- 30(i.a.) PCC 76 78 (4-methoxybenzyl)-N-methyl- PAC 23 596,7-dihydro-5H-pyrrolo[1,2- c]imidazole-5-carboxamide entantiomer B**(Example 9-9 as described in WO2007/024945)4-(6,7-Dihydro-5H-pyrrolo[1,2- 10 (p.o.) PCC 24 30 c]imidazol-5-yl)-3-PAC 65 72 fluorobenzonitrile enantiomer B** ((R) enantiomer, asdescribed in Exampe 3, in WO2007/024945) **Please refer to WO2007/024945for details.

Example 7

A pilot, single-blind, forced-titration study was performed to assessthe hormonal effects of a compound of formula (I),4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate, in patients with diagnosed primaryhyperaldosteronism. The clinical study was designed, implemented andreported in accordance with the ICH harmonized tripartite guidelines forgood clinical practice with applicable local regulations. Each patientparticipated in a screening/washout period, a 2-week placebo run-inperiod, a 4-week treatment period, and a 1-week placebo wash-out. Thetreatment period consisted of the oral administration of compound offormula (I) twice daily at a dose of 0.5 mg for two weeks followed by adose increase to 1.0 mg twice daily for another 2 weeks. Blood sampleswere taken at baseline, day 1 and 2, day 8, day 15, day 22, day 29 and30 (all at predose i.e., 12 h after the last dose), and at the finalstudy day 36. Each sample was assessed for aldosterone andimmunoreactive active renin, 11-deoxycorticosterone, cortisol,11-deoxycortisol and adrenocorticotropin (ACTH) after subjects were atrest for at least 60 minutes to avoid any postural or stress-inducedvalue changes. Plasma aldosterone was measured using a commerciallyavailable radioimmunoassay kit (DPC, France). Plasma Active Renin wasmeasured using the two monoclonal antibodies 3E8 and 125I-4G1 in acommercially available immunoradiometric kit (CisBio, France). Plasma11-deoxycorticosterone, cortisol and 11-deoxycortisol were measuredusing a standardized LC-MS/MS method. Plasma ACTH was measured using acommercially available immunoradiometric kit (CisBio, France).

Statistical analysis of the pharmacodynamic biomarker were summarizedusing descriptive statistics as well as graphical and/or regressionmethods. The administration of a compound of formula (I),4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate to patients with primary hyperaldosteronism over a2-times 2-week period is shown in FIG. 2 and elicited as anticipatedpotent aldosterone suppression that was reflected by an increased activerenin level but also by an unexpectedly high build-up of the precursorsteroid 11-deoxycorticosterone. The accumulation of11-deoxycorticosterone (P11DOCS) was stimulated by increased ACTHlevels. The increased adrenocorticotropin levels resulted frominhibition of cortisol synthesis via 11-beta-hydroxylase as reflected bydecreasing cortisol levels and an accumulation of the enzyme substrate11-deoxycortisol (P11DOC). The increased 11-deoxycorticosterone levelsin presence of inhibited stress hormone synthesis are shifted towardsincreased synthesis of the adrenal androgens androstendione anddehydroepiandrosterone (see FIG. 1, a diagram for adrenalsteroidogenesis). Thus, the compound of formula (I), exhibited thepharmacological profile of a pleiotropic adrenal hormone-modifying agentas it inhibits the synthesis of both, aldosterone and cortisol, while itincreases the levels of ACTH, 11-deoxycorticosterone and finally thesynthesis of the adrenal androgens, androstendione anddehydroepiandrosterone.

Example 8

An open-label, single arm, sequential dose-escalation, multi-centerstudy in patients with Cushing's disease is performed as describedherein below.

The study consists of a 10-14-day baseline period, a 10-week treatmentperiod consisting of biweekly treatment with escalating doses and a14-day washout period followed by a Study Completion evaluation at 14days after the last drug administration. The study drug is applied atescalating doses of 2 mg, 5 mg, 10 mg, 20 mg and 50 mg twice daily (bid)each for a two week period (see study timeline below). The optimaltherapeutic dose is dependent on the severity and responsiveness of theunderlying pathological condition.

Population:

The study population is comprised of male and female patients withendogenous hypercortisolism due to increased ACTH [AdrenocorticotropicHormone] production from the pituitary (Cushing's disease).

Male or female patients aged 18-75 years

Patients have confirmed Cushing's Disease as evidenced by:

-   -   UFC [Urinary Free Cortisol]>1.5×ULN [Upper Limit of Normal]        (Mean value of three 24-hour urine samples collected within 14        days)    -   Morning plasma ACTH above 10 pg/mL

Subjects are permitted to washout current drug therapy to meet theseentry criteria if they have a known diagnosis of Cushing's disease.

Therapy:

Subjects start on a dose of 2 mg b.i.d. of4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate and increase their dose every 2 weeks. The optimaltherapeutic dose is determined by the treatment effect and tolerabilityof the intervention.

Efficacy/Pharmacodynamic Assessments:

Efficacy assessments include urinary free cortisol, plasma ACTH,cortisol and renin, plasma and urine aldosterone, plasma and urinesodium and potassium, salivary cortisol and aldosterone and plasmainsulin.

Safety Assessments:

Safety assessments include physical examinations, ECGs(Electrocardiograms), vital signs, standard clinical laboratoryevaluations (hematology, blood chemistry, urinalysis,) adverse event andserious adverse event monitoring.

Data Analysis:

The primary efficacy variable is defined as the proportion of respondersto4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate. A patient is considered to be a responder if themean UFC level from the Week 10 24-hour urine samples is ≦1×ULN.Patients who discontinue for a disease or treatment related reason (e.g.death, adverse event, clinical disease progression etc.), or whose meanWeek 10 24-hour UFC levels are higher than the normal limit areclassified as non-responders. Patients who have only one baseline orpost-baseline 24-hour UFC measurement will not be included in theprimary efficacy analyses.

1. A method of treating a disease or disorder characterised by increasedstress hormone levels and/or decreased androgen hormone levels in asubject, comprising administering to the subject a therapeuticallyeffective amount of a compound represented by formula (I):

wherein n is 1 or 3; R is hydrogen or —C(O)N(R_(a))(R_(b)) wherein R_(a)and R_(b) are independently —(C₁-C₄)alkyl, or —(C₁-C₄)alkyl-(C₅-C₇)aryl,wherein each of R_(a) and R_(b) is optionally substituted by—(C₁-C₄)alkoxy; R₁, R₂, and R₃, are independently hydrogen, halogen,cyano or —(C₆-C₁₀)aryl, wherein said —(C₆-C₁₀)aryl is optionallysubstituted by halogen, with the proviso that no more than one of R₁,R₂, and R₃ is hydrogen; and R₄ and R₅ are hydrogen; or apharmaceutically acceptable salt thereof.
 2. The method according toclaim 1, wherein compound is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitrilehaving formula


3. The method according to claim 1, wherein compound is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate.
 4. The method according to claim 1, wherein thedisease or disorder is heart failure, cachexia, acute coronary syndrome,chronic stress syndrome, cushing's syndrome, metabolic syndrome orhypercortisolemia.
 5. The method according to claim 4, wherein thedisease or disorder is selected from: acute heart failure, acutedecompensated heart failure, chronic heart failure, chronic heartfailure with impaired exercise tolerance, chronic heart failure withmuscle weakness, cardiac cachexia, COPD-induced cachexia,cirrhosis-induced cachexia, tumor-induced cachexia, or viral(HIV)-induced cachexia.
 6. A method of treating heart failure, cachexia,acute coronary syndrome, chronic stress cushing's syndrome, metabolicsyndrome or hypercortisolemia, comprising administering to the subject atherapeutically effective amount of a compound represented by formula(I):

wherein n is 1 or 3; R is hydrogen or —C(O)N(R_(a))(R_(b)) wherein R_(a)and R_(b) are independently —(C₁-C₄)alkyl, or —(C₁-C₄)alkyl-(C₅-C₇)aryl,wherein each of R_(a) and R_(b) is optionally substituted by—(C₁-C₄)alkoxy; R₁, R₂, and R₃, are independently hydrogen, halogen,cyano or —(C₆-C₁₀)aryl, wherein said —(C₆-C₁₀)aryl is optionallysubstituted by halogen, with the proviso that no more than one of R₁,R₂, and R₃ is hydrogen; and R₄ and R₅ are hydrogen; or apharmaceutically acceptable salt thereof.
 7. The method according toclaim 6, wherein the compound is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitrile.8. The method according to claim 6, wherein the compound is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate.
 9. The method according to claim 6, wherein thedisease or disorder is selected from: acute heart failure, acutedecompensated heart failure, chronic heart failure, chronic heartfailure with impaired exercise tolerance, chronic heart failure withmuscle weakness, cardiac cachexia, COPD-induced cachexia,cirrhosis-induced cachexia, tumor-induced cachexia, or viral(HIV)-induced cachexia. 10-13. (canceled)
 14. A compound of formula (I)

wherein n is 1 or 3; R is hydrogen or —C(O)N(R_(a))(R_(b)) wherein R_(a)and R_(b) are independently —(C₁-C₄)alkyl, or —(C₁-C₄)alkyl-(C₅-C₇)aryl,wherein each of R_(a) and R_(b) is optionally substituted by—(C₁-C₄)alkoxy; R₁, R₂, and R₃, are independently hydrogen, halogen,cyano or —(C₆-C₁₀)aryl, wherein said —(C₆-C₁₀)aryl is optionallysubstituted by halogen, with the proviso that no more than one of R₁,R₂, and R₃ is hydrogen; and R₄ and R₅ are hydrogen; or apharmaceutically acceptable salt thereof, for use in the treatment of adisorder or disease characterised by increased stress hormone levelsand/or decreased androgen hormone levels in a subject.
 15. A compoundaccording to claim 14, wherein the compound of formula (I) is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitrile.16. A compound according to claim 14, wherein the compound of formula(I) is4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluororbenzonitriledihydrogen phosphate.
 17. A compound according to claim 14, wherein thedisease or disorder is heart failure, cachexia, acute coronary syndrome,chronic stress syndrome, cushing's syndrome, metabolic syndrome orhypercortisolemia.
 18. A compound according to claim 16, wherein thedisease or disorder is cushing's syndrome.
 19. A compound according toclaim 17, wherein the disease or disorder is selected from: acute heartfailure, acute decompensated heart failure, chronic heart failure,chronic heart failure with impaired exercise tolerance, chronic heartfailure with muscle weakness, cardiac cachexia, COPD-induced cachexia,cirrhosis-induced cachexia, tumor-induced cachexia, or viral(HIV)-induced cachexia.